Immunological detection of phosphorylation.

Incorporation of phosphates into serine, threonine, and tyrosine acceptors in proteins is a common mechanism for regulating protein function. This unit presents protocols that use specific anti-phosphoamino acid (PAA) and anti-phosphoprotein antibodies to detect protein phosphorylation and protein kinase activity. Immunoblotting to detect protein phosphorylation using either anti-PAA or anti-phosphoprotein antibodies. This is a convenient method that usually yields impressive results. Phosphorylation can also be detected by immunoprecipitation followed by immunoblot analysis or by immunofluorescent staining; these methods are typically more complicated and time consuming. All three methods have been successfully used to detect protein phosphorylation with a wide variety of antibodies and most phosphorylated proteins.